In ovo vaccination of marek&#39;s disease type I virus

ABSTRACT

The preset invention provides a method for immunizing chickens which comprises inoculating into growing egg a composition comprising either cell-free attenuated viruses of Marek&#39;s disease type 1 or cells infected with attenuated viruses of Marek&#39;s disease type 1 capable of producing cell-free viruses. The present invention also provides a method for immunizing chickens which comprises inoculating into growing egg a mixed vaccine comprising said composition plus another vaccine from at least one microorganisms selected from the group consisting of viruses other than virus of Marek&#39;s disease type 1, bacteria and protozoan.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/JP99/06866 which has an Internationalfiling date of Feb. 8, 1999, which designated the United States ofAmerica.

The present invention relates to a prophylaxis for chicken Marek'sdisease induced by Marek's disease type I virus (hereinafter alsoreferred to as “MDV-1”). More specifically, the present inventionrelates to a method for immunizing chickens comprising inoculating intoa growing chicken egg either a composition comprising cell-freeattenuated live MDV-1 or a composition comprising cells infected withattenuated live MDV-1 capable of producing cell-free viruses.Furthermore, the present invention provides a method for efficientlyimmunizing chickens against plural diseases with recombinant MDV-1.

BACKGROUND OF THE INVENTION

Marek's disease is a chicken infectious disease characterized byinfiltration or tumorous proliferation of lymphoids by MDV-1 associatedwith symptoms such as leg paralysis, formation of lymphoma in variousorgans of viscera, weight loss, anemia, diarrhea, etc. In the field,Marek's disease leads chicken to death or partial or total disposal in apoultry laboratory and hence results in much economic loss in poultryfarmer.

For protection from Marek's disease, attenuated live vaccine hashitherto been used, i.e. MDV-1, MDV-2 or herpes virus of turkey (HVT,MDV-3) alone or in admixture has been subcutaneously inoculated intonewborn chicken. In such a case, a dosage form of virus-infected cellswas usually employed. However, it was reported that as to Marek'sdisease virus with extremely high pathogenicity such as very virulentstrain or very virulent (+) as reported in recent years, MDV-2 or HVTexhibits poor effectiveness but only vaccine of MDV-1 can effectively beused (R. L. Witter, AVIAN DISEASE 41: 149-163, 1997).

Nowadays, a number of vaccines have been developed for various chickeninfectious diseases. Prophylaxis of diseases by vaccination is a rulingmeasure for sanitation in the field of poultry irrespective of breedingchicken, laying hen or chicken for meat. On the other hand, labor costdue to scale-up of poultry or crammed schedule of frequent inoculationof vaccine is a burden to poultry farmer. For obviating this,development of more effective and efficient vaccination such asreduction in frequency of inoculation by mixing several vaccines (i.e.mixed vaccine) or improvement of vaccination has been attempted.

As one of efficient way of vaccinations, Sharma et al. reportedvaccination into growing chicken egg (“in ovo” inoculation) (J. M.Sharma et al., AVIAN DISEASE 26(1): 134-149, 1982). That is, accordingto Sharma et al., chicken hatched from a growing chicken egg inoculatedwith HVT-infected cells and chicken inoculated with said HVT afterhatching were attacked by Marek's disease virus (hereinafter alsoreferred to as “MDV”), and as a result, it was found that the chickenfrom vaccinated growing egg exhibited more potent resistance against theattack as compared to the chicken vaccinated after hatching,demonstrating that in ovo inoculation can effectively be used forimmunization.

Reddy et al. prepared a recombinant HVT (rHVT) wherein a fusion protein(F protein) gene of Newcastle disease virus (hereinafter also referredto as “NDV”) and hemagglutinin-neuraminidase (HN) glycoprotein gene ofNDV were incorporated into HVT genome (S. K. Reddy et al., Vaccine14(6): 469-477, 1996). They inoculated cells infected with this virusinto growing chicken egg on day 18. After hatching, the chicken wasattacked by both NDV and MDV viruses to thereby demonstrate that therecombinant virus could effectively protect the chicken from the attackof both viruses. The effect of in ovo vaccination has also beenestimated for an inactivated oil-emulsified vaccine of NDV and avianinfluenza virus (H. Stone, AVIAN DISEASE 41: 856-863, 1997) or for liveEimeria sporozoite vaccine (Evans et al., WO 96/40233).

Chicken immunization at the stage of growing egg as described above,called “in ovo vaccination”, can be expected to eliminate or reducelabor and cost and hence an automated device for inoculation intochicken egg becomes prevailing.

However, Sharma et al. reported that MDV-1-infected cells wereinoculated into growing chicken egg and growth of said virus in chickenembryo tissue was investigated and, as a result, no viral growth wasobserved (J. M. Sharma et al., AVIAN DISEASES 31: 570-576, 1987). Theyalso reported that chicken hatched from said growing egg was notprotected from the attack of virulent MDV strain. This result suggeststhat in ovo vaccination against Marek's disease with MDV-1 is not soeasy. Accordingly, it is the present situation that a univalent or mixedvaccine of HVT or MDV-2 SB-1 strain has alternatively been used as inplace of MDV-1, a causing virus of Marek's disease.

On the other hand, there is a report of attempting to inoculate MDV-1,having been regarded as being ineffective for in ovo vaccination, intogrowing chicken egg (Taniguchi et al., 117th Japan Veterinary ScienceAssociation excerpt, p.198, 1994, Tokyo). In this case, if inoculationis made directly to fetus, the virus efficiently grew and successfulimmunization results. However, indeed, failure in direct inoculation tofetus frequently occurs. In spite of such circumstances, fieldapplication has been conducted because even if some individuals failedto be immunized, contact infection occurring about two weeks after MDV-1inoculation would impart immunization to those individuals that failedto be immunized.

However, if non-immunized chicken is exposed to virulent MDV strain inthe field after hatching and prior to infection with said MDV-1 vaccineby contact infection, then said chicken cannot be protected from Marek'sdisease and hence considerable economic loss will results. Thus, forthorough protection from Marek's disease, it is most important whetheror not immunization is established within a week just after hatching.Accordingly, if possible, it is desirable to surely infect fetus in thegrowing egg with vaccine virus.

There is also a care that direct inoculation of needle into fetus mightpossibly damage the fetus badly in some cases.

Under the circumstances, there is a need to develop a more effectiveMDV-1 vaccine for Marek's disease that is applicable for in ovovaccination.

DISCLOSURE OF THE INVENTION

The present inventors thoroughly investigated in order to develop a moreeffective MDV-1 vaccine for Marek's disease that is applicable for inovo vaccination. As a result, it was found that a cell-free viralsolution prepared from a solution of ruptured cells infected with eitherattenuated MDV-1 or recombinant MDV-1 was inoculated into growing egg tothereby exhibit viral growth. It was also found that antibodies to MDV-1and to other viral antigens incorporated into MDV-1 were induced inserum of chicken that hatched from said virus-inoculated growing egg. Assuch, the present inventors have completed the present invention.

That is, an object of the present invention is to provide a method forimmunizing chickens which comprises inoculating into growing egg acomposition comprising either cell-free attenuated live MDV-1 or cellsinfected with attenuated live MDV-1 capable of secreting cell-freeattenuated live MDV-1 out of the cells. The present invention alsoprovides a method for efficiently immunizing chickens against pluraldiseases by using a recombinant MDV-1 in the above method forimmunization.

Another object of the present invention is to provide a method forimmunizing chickens which comprises inoculating into growing egg a mixedvaccine comprising the above cell-free MDV-1 plus another vaccinederived from at least one microorganisms selected from the groupconsisting of viruses other than MDV-1, bacteria and protozoan.

The method of the present invention is characterized by a compositioncomprising cell-free attenuated live MDV-1, a process for preparing thesame and in ovo vaccination of said composition.

BEST MODE FOR CARRYING OUT THE INVENTION

The term “cell-free virus(es)” as used herein means viruses that arepresent in the state of being free from cells.

For preparing a composition comprising cell-free attenuated live MDV-1,attenuated MDV-1 viruses are infected to host cells and grown therein.The virus-infected cells are then collected by centrifugation at a lowspeed. After addition of a buffer supplemented with an appropriateamount of sugars, the cells are ruptured by sonication orfreezing-thawing or by physically rupturing the cells. MDV-1 viruses arethen extracted from the obtained solution of the ruptured cells andpurified. For the purpose of the present invention, centrifugation at alow speed is carried out at 1,000 to 3,000 rpm for 3 to 10 minutes usingKUBOTA, KN-30F or another centrifuging machine with equivalent turningradius. Alternatively, the obtained solution of ruptured cells maydirectly be used as it stands without any treatment.

Attenuated MDV-1 includes CVI-988 strain, 61-554 strain (Sakaguchi etal., Japanese Patent Publication No. 6-22757), Md11/75C (R. L. Witter,AVIAN DISEASE 31: 752-765, 1987) and the like as well as recombinantviruses derived therefrom.

The composition of the present invention may also be prepared by usingattenuated MDV-1-infected cells capable of secreting cell-free virusesout of the cells. Such attenuated MDV-1-infected cells capable ofsecreting cell-free viruses out of the cells may be prepared byrepeatedly culturing and growing cell-free attenuated MDV-1 occurring ina trace amount in supernatant of said virus-infected cells as describedin more detail in Sakaguchi et al., Japanese Patent Publication No.6-22757.

As a host cell for viral infection, any culture cell may be used as faras attenuated MDV-1 can grow therein and it does not produce contaminateviruses. Preferably, avian-derived culture cells are used. Chickenembryo fibroblast cells (CEF cells), duck embryo fibroblast cells,chicken embryo-derived cell strain CHCC-OU2 (Ogura, H. et al., Acta MedOkayama 41(3): 141-143, 1987, and Coussens et al., Japanese PatentPublication No. 9-173059), quail-derived cell strain QT-35 (Spijkers etal., Japanese Patent Publication No. 9-98778) and the like are used withchicken embryo fibroblast cells (CEF cells) being preferred.

A culture medium for culturing host cells includes a medium commonlyused for tissue culture such as M199-earle base (Nissui), Eagle MEM(E-MEM) (Nissui), Dulbecco MEM (D-MEM) (Nissui), SC-UCM102 (Nissui),UP-SFM (GIBCO BRL), EX-CELL302 (Nichirei), EX-CELL293-S (Nichirei),TFBM-01 (Nichirei), ASF104, etc. These culture media are used withsupplement of amino acids, salts, anti-fungal or anti-bacterial agent,animal serum and the like. They may optionally be used as a serum-freemedium by not supplementing serum.

Cells or virus-infected cells are cultured under normal conditions. Thatis, culture temperature and period may appropriately be adjusteddepending on various factors such as types of cells, inoculation amountof viruses and scale and process of culture or a combination thereof,culture temperature may range from 35° C. to 41° C., preferably from 37°C. to 38° C. and culture period may range from 2 to 7 days, preferablyfrom 3 to 4 days.

The thus prepared composition of the present invention is inoculatedinto growing chicken egg. The term “growing chicken egg” as used hereinmeans a fertilized egg that is incubated and in the progress until 21days after fertilization when chicken is hatched. Vaccination forMarek's disease may be carried out after incubation for 17 to 19 days.

The composition of the present invention may be inoculated into growingchicken egg by, for example, injecting an inoculation needle (e.g.24G1·¼ needle) into growing chicken egg of 18 days old at an air chamber(i.e. at an obtuse-angled site of egg) to introduce viral solution.

In accordance with the composition of the present invention comprisingeither cell-free attenuated MDV-1 or attenuated MDV-1-infected cellscapable of producing cell-free viruses, attenuated MDV-1 can efficientlyinfect in the growing egg and induce immunization for Marek's disease.

The composition of the present invention may also be used for in ovovaccination as a mixed vaccine in combination with at least one vaccineselected from the group consisting of vaccines to other viruses such ase.g. avian infectious bronchitis virus, avian infectious bursal diseasevirus, avian encephalomyelitis virus, egg drop syndrome virus, influenzavirus, reovirus, adenovirus, hydropericardium syndrome virus, etc.;bacteria such as e.g. Haemophilus paragallinarum, Salmonellatyphimurium, S. enteritidis, S. pullorl, S. gallinarum, S. choleraesuis,E. coli, Clostridium spp., Campylobacter spp., Mycoplasma spp.,enterococcus, etc.; and protozoan such as e.g. Leucocytozoon caulleryi,Eimeria tenella, E. maxima, E. acervulina, E. brunetti, E. necatrix,chicken malaria, etc.

Infection-protecting antigen expressed by the recombinant Marek'sdisease virus used in the present invention includes, in addition to Fprotein of Newcastle disease virus exemplified above, HN protein ofNewcastle disease virus, core protein, capsid protein or glycoprotein ofvarious viruses (e.g. spike protein or nucleocapsid protein of avianinfectious bronchitis virus, VP2 of avian infectious bursal diseasevirus (IBDV), capsid protein of avian encephalomyelitis, capsid proteinof egg drop syndrome virus, VP1+VP2 of avian anemia virus, glycoproteinB of avian infectious laryngotracheitis, membrane protein or gag ofavian leukemia virus or reticuloendotheliosis, glycoprotein ofrhinotracheitis of turkeys virus, HA protein of avian pox virus, HA ofinfluenza virus, capsid protein of avian reovirus, capsid protein ofavian adenovirus, etc.), cilia, flagella, toxins, hemolysins, membraneproteins such as porin, peptides with antigenicity of O-antigen ofbacteria (e.g. Haemophilus paragarinarum, Salmonella choleraesuis, E.coli, Campylobacter, Clostridium, Mycoplasma, enterococcus, etc.), andproteins or glycoproteins from protozoan (e.g. Leucocytozoon caulleryi,Eimeria tenella, E. maxima, E. acervulina, E. brunetti, E. necatrix,avian malaria), and the like.

Such recombinant Marek's disease virus can be prepared using an ordinaryprocess for preparing a recombinant virus well known in the art.

Other than ND, antigens that were actually incorporated into arecombinant MDV-1 and reported to be immunogenic in chicken includesIBDV-VP2 antigen as reported by Tsukamoto et al., Virology 257: 352-362,1999. A plural of recombinant viruses incorporating marker gene, lacZ,were also reported (Sakaguchi et al., Virology 195: 140-148, 1993;Percells et al., J. Virology 68: 8239-8253, 1994; Schat et al., J. gen.Virology 70: 841-849, 1998).

The present invention is explained in more detail by means of thefollowing Examples, but should not be construed to be limited thereto.

EXAMPLE EXAMPLE 1 In Ovo Vaccination of MDV-1

(1) Preparation of Materials for Inoculation of Growing Chicken egg

About 1×10⁸ CEF: cells and about 1×10⁶ PFU of MDV-1 Rispens strain weresuspended in E-MEM medium (40 ml) supplemented with 5% fetal bovineserum (FBS). The suspension was placed in a 175 cm² culture flask andincubated at 37° C. for 4 days. When 80% or more cytopathic effect (CPE)was observed, virus-infected cells were collected in a usual manneremploying 0.1% EDTA-0.125% trypsin (DIFCO). The collected cells weresuspended in 40 ml of E-MEM medium. From this suspension, materials forinoculation into growing chicken egg were prepared by the following twoprocesses:

(a) A solution of Virus-infected Cells

The above suspension (1 ml) was serially diluted 10-fold with E-MEM toprepare a solution of virus-infected cells.

(b) A Solution of Cell-free Viruses

The remaining suspension (39 ml) was centrifuged at a low speed at 1,500rpm for 5 minutes. After removing supernatant by suction, the cells weresuspended by adding 2 ml of SPGA-SBT solution (prepared in accordancewith B. W. Calnek et al., Appl. Microbiol. (20): 723-726, 1970; B. R.Cho, Avian Dis. 22 (1): 170-176, 1977; 0.218 M sucrose, 0.0038 Mpotassium dihydrogenphosphate, 0.0072 M dipotassium hydrogenphosphate,0.0049 M sodium glutamate, 1% bovine serum albumin, 10% sorbitol). Thesuspension was sonicated with TOMY SEICO Co., LTD, Handy sonic UR-20P,Power cont. 4 for 1 minute and then centrifuged at a low speed at 2,500rpm for 5 minutes. The obtained supernatant was serially diluted 10-foldto prepare a solution of cell-free viruses.

Viruses in the inoculation materials were counted as follows: CEF cellspreviously cultured were collected with EDTA-trypsin and centrifuged at1,500 rpm for 5 minutes. The obtained cellular sediment was againsuspended in 5%-FBS at a concentration of 6×10⁵ cells/ml (hereinafterreferred to as “CEF2nd”).

The CEF2nd cells, (9×10⁶ cells/15 ml in a 10 cm Petri dish) werecultured for 4 hours and inoculated with 1 ml of a vaccine solution ofeach dilution by tilting every 20 minutes for 1 hour. The cells werethen added with E-MEM medium (5 ml) and incubated in CO₂, incubator at37° C. overnight. The next day, E-MEM supplemented with 2% methylcellulose (Sigma) and 1% FCS was overlaid, the cells were incubated inCO₂ incubator at 37° C. for 10 days and the number of plaques appearedwas counted.

(2) Inoculation into Growing Chicken Egg

Each group consisted of six growing chicken eggs of 18 days old (SPFmanufactured by Juridical Foundation The Chemo-Sero-Therapeutic ResearchInstitute). Each 0.1 ml of the solution of virus-infected cells seriallydiluted 10-fold or of the solution of cell-free viruses was inoculatedinto the egg by injecting 24G1·¼ inoculating needle (Nipro) at an airchamber (an obtuse angled site) of the egg with about 2.5 cm depth. Theeggs were incubated at 37° C. for 3 days. A week after hatching, 1 ml ofblood was drawn from the heart with a heparinized 5 ml disposablesyringe (Nipro) and 21G 1 inch needle (Nipro). A fraction of mononuclearcells was separated from said blood using Ficoll Paque Plus (Pharmacia)in. accordance with a manufacture's instruction. A total of thisfraction was inoculated into CEF2nd cells (9×10⁶ cells/15 ml, in Petridish of 10 cm diameter) previously cultured for 4 hours. Ten days later,plaques of Marek's disease viruses appeared were counted. Recovery ratesof viruses in the group of inoculation with the solution ofvirus-infected cells and in the group of inoculation with the solutionof cell-free viruses are shown in Tables 1 and 2, respectively. Amonghatched chicken, blood was drawn from each 5 chicken in the groups toconduct viral recovery.

TABLE 1 Inoculated amount Rate of viral infection per chicken (PFU)(Positive No./Total No.) 1280 4/5 128 2/5 12.8 0/5 1.28 0/5

TABLE 2 Inoculated amount Rate of viral infection per chicken (PFU)(Positive No./Total No.) 540 5/5 54 5/5 5.4 5/5 0.54 NT

As apparent from Tables 1 and 2, viruses were recovered from merely lessthan the half of the individuals among chicken from eggs inoculated with128 PFU in the group of inoculation with the solution of virus-infectedcells. On the contrary, in the group 2 inoculated with the solution ofcell-free viruses, viruses were recovered from all the five chicken evenin the group inoculated with as low as 5.4 PFU. This demonstrates thatMDV-1 viruses indeed propagate in case of the solution of cell-freeviruses and hence the solution of cell-free viruses can effectively beused.

EXAMPLE 2 In Ovo Vaccination of Recombinant MDV-1

(1) Preparation of Materials for Inoculation of Growing Chicken Egg

About 1×10⁸ CEF cells and about 1×10⁶ PFU of recombinant virus rMDV1US10P(F) strain wherein a gene for Newcastle disease virus F (NDV-F)protein was incorporated (Sonoda et al., Current research on Marek'sdisease, p.408, 1996) were suspended in E-MEM medium (40 ml)supplemented with 5% fetal bovine serum (FBS). The suspension was placedin a 175 cm² culture flask and incubated at 37° C. for 4 days. When 80%or more cytopathic effect (CPE) was observed, virus-infected cells werecollected in a usual manner employing 0.1% EDTA-0.125% trypsin (DIFCO).The collected cells were suspended in 10 ml of E-MEM medium. From thissuspension, materials for inoculation into growing chicken egg wereprepared by the following processes:

Preparation of a Solution of Cell-free Viruses

As described in Example 1 (1), the suspension (40 ml) was centrifuged ata low speed at 1,500 rpm for 5 minutes. After removing supernatant bysuction, the cells were suspended by adding 2 ml of SPGA-SBT solution.The suspension was sonicated for 1 minute and then centrifuged at a lowspeed at 2,500 rpm for 5 minutes. The obtained supernatant was seriallydiluted 10-fold to prepare a solution of cell-free viruses.

As described in Example 1(1), viruses in the inoculation materials werecounted as follows: i.e. the CEF2nd cells (9×10⁶ cells/15 ml in a Petridish of 10 cm diameter) were prepared as described in Example 1 (1) andpreviously cultured for 4 hours. The cells were inoculated with 1 ml ofa vaccine solution of each dilution by tilting every 20 minutes for 1hour. The cells were then added with E-MEM medium (5 ml) and incubatedin CO₂ incubator at 37° C. overnight. The next day, E-MEM supplementedwith 2% methyl cellulose (Sigma) and 1% FCS was overlaid, the cells wereincubated in CO₂ incubator at 37° C. for 10 days and the number ofplaques appeared was counted.

(2) Inoculation into Growing Chicken Egg

Each group consisted of five growing chicken eggs of 18 days old (SPFmanufactured by Juridical Foundation The Chemo-Sero-Therapeutic ResearchInstitute). Each 0.1 ml of the solution of virus-infected cells seriallydiluted 10-fold or of the solution of cell-free viruses was inoculatedinto the egg by injecting 24G1·¼ inoculating needle (Nipro) at an airchamber (an obtuse angled site) of the egg with about 2.5 cm depth. Theeggs were incubated at 37° C. for 3 days. A week after hatching, 1 ml ofblood was drawn from the heart with a heparinized 5 ml disposablesyringe (Nipro) and 21G 1 inch needle (Nipro). A fraction of mononuclearcells was separated from said blood using Ficoll Paque Plus (Pharmacia)in accordance with a manufacture's instruction. A total of this fractionwas inoculated into CEF2nd cells (9×10⁶ cells/15 ml, in Petri dish of 10cm diameter) previously cultured for 4 hours. Ten days later, plaques ofMarek's disease virus appeared were counted.

TABLE 3 Inoculated amount Rate of viral infection Average per chicken(PFU) (Positive No./Total No.) Viremia¹⁾ 320 5/5 3.27 32 5/5 1.82 3.23/5 1.61 0.32 0/5 NT ¹⁾Viral amount per 1 × 10⁶ mononuclear cells

As apparent from Table 3, viruses were recovered from all the fivechicken even in the group inoculated with as low as 32 PFU. Thus,effectiveness of the solution of cell-free viruses was confirmed in caseof recombinant MDV-1 virus.

(3) Antibody Test

Growing chicken eggs were inoculated as described above except that eachgroup consisted of six growing chicken eggs of 18 days old (SPFmanufactured by Juridical Foundation The Chemo-Serb-Therapeutic ResearchInstitute). When chicken from the growing eggs became 8 weeks old, bloodwas drawn and an antibody to NDV-F protein was determined.

The antibody was detected by ELISA with NDV-F expressing cells as anantigen. Detail of this procedure is described in Sakaguchi M. et al.,Vaccine, 1996 June, 14(8): 747-52. Rates of NDV-F antibody positiveindividuals in the group of inoculation with the solution ofvirus-infected cells and in the group of inoculation with the solutionof cell-free viruses are shown in Tables 4 and 5, respectively.

TABLE 4 Inoculated amount Rate of NDV-F Ab positive individuals perchicken (PFU) (Positive No./Total No.) 84 2/6 8.4 0/6

TABLE 5 Inoculated amount Rate of NDV-F Ab positive individuals perchicken (PFU) (Positive No./Total No.) 70  5/5 7 4/6   0.7 2/6 Noinoculation 0/6

As shown in Table 5, all the chickens were positive for the antibody inthe group of chickens from the eggs inoculated with 70 PFU cell-freeviruses. In the group of chickens from the eggs inoculated with 7 PFUcell-free viruses, four among six chickens were positive. On thecontrary, in the group of chickens inoculated with the solution ofvirus-infected cells, only two among six chickens were found to bepositive when 84 PFU was inoculated. Thus, the cell-free virusinoculated group showed a higher positive conversion even with a loweramount of viruses by 10-fold than the virus-infected cells group,demonstrating usefulness of the cell-free viruses for immunization ofgrowing egg in view of antibody response.

INDUSTRIAL APPLICABILITY

In accordance with the present invention, there are provided MDV-1 livevaccine for prophylaxis of chicken Marek's disease and a method forimmunization by in ovo inoculation using said live vaccine. The methodof the present invention allows for protection from Marek's diseasevirus infection more efficiently and effectively as compared to theconventional methods. Furthermore, the method of the present inventionalso allows for immunization for foreign gene products simultaneouslyexpressed by MDV-1 in a laborsaving manner.

All the disclosures of all publications (including patents, patentapplications, periodical publications, laboratory manuals, books andother publications) recited herein are incorporated herein forreference.

What is claimed is:
 1. A method for immunizing chickens which comprisesinoculating into a growing egg a composition comprising either cell-freeattenuated live viruses of Marek's disease type 1 or cells infected withattenuated live viruses of Marek's disease type 1 capable of producingcell-free viruses.
 2. The method of claim 1 wherein said attenuatedviruses of Marek's disease type 1 are a recombinant virus of Marek'sdisease type
 1. 3. The method of claim 2 wherein genes incorporated intosaid recombinant virus of Marek's disease type 1 code for antigens fromviruses other than Marek's disease type 1 virus, bacteria or protozoa.4. The method of claim 3 wherein said antigens are selected from thegroup consisting of F protein of Newcastle disease virus, HN protein ofNewcastle disease virus, spike protein of avian infectious bronchitisvirus, core protein of avian infectious bronchitis virus, VP2 of avianinfectious bursal disease, virus (IBDV), capsid protein of avianencephalomyelitis, capsid protein of egg drop syndrome virus, VP1+VP2 ofavian anemia virus, glycoprotein B of avian infectiouslaryngotracheitis, membrane protein of avian leukemia virus ofreticuloendotheliosis, gag of avian leukemia virus ofreticuloendotheliosis, glycoprotein of rhinotracheitis of turkeys virus,HA protein of avian pox virus, HA of influenza virus, capsid protein ofavian reovirus, capsid protein of avian adenovirus, cilia, flagella,toxins, hemolysins, membrane proteins, peptides with antigenicity ofO-antigen and proteins of glycoproteins from protozoa.
 5. A method forimmunizing chickens which comprises inoculating into a growing egg amixed vaccine comprising either cell-free attenuated viruses of Marek'sdisease type I or cells infected with attenuated viruses of Marek'sdisease type 1 capable of producing cell-free viruses plus anothervaccine derived from at least one microorganism selected from the groupconsisting of viruses other than MDV-1, bacteria and protozoa.
 6. Themethod of claim 5 wherein said microorganisms are selected from thegroup consisting of avian infectious bronchitis virus, avian infectiousbursal disease virus, avian encephalomyelitis virus, egg drop syndromevirus, influenza virus, reovirus, adenovirus, hydropericardium syndromevirus, bacteria and protozoan.
 7. The method of claim 5 or 6 whereinsaid microorganisms are not inactivated.
 8. A method for immunizingchickens which comprises inoculating into a growing egg a compositioncomprising cell-free attenuated live viruses of Marek's disease type 1.9. The method of claim 8 wherein said attenuated viruses of Marek'sdisease type 1 are a recombinant virus of Marek's disease type
 1. 10.The method of claim 9 wherein genes incorporated into said recombinantvirus of Marek's disease type 1 code for antigens from viruses otherthan Marek's disease type 1 virus, bacteria or protozoa.
 11. The methodof claim 10 wherein said antigens are selected from the group consistingof F protein of Newcastle disease virus, HN protein of Newcastle diseasevirus, spike protein of avian infectious bronchitis virus, core proteinof avian infectious bronchitis virus, VP2 of avian infectious bursaldisease virus (IBDV), capsid protein of avian encephalomyelitis, capsidprotein of egg drop syndrome virus, VP1+VP2 of avian anemia virus,glycoprotein B of avian infectious laryngotracheitis, membrane proteinof avian leukemia virus of reticuloendotheliosis, gag of avian leukemiavirus of reticuloendotheliosis, glycoprotein of rhinotracheitis ofturkeys virus, HA protein of avian pox virus, HA of influenza virus,capsid protein of avian reovirus, capsid protein of avian adenovirus,cilia, flagella, toxins, hemolysins, membrane proteins, peptides withantigenicity of O-antigen and proteins of glycoproteins from protozoa.